The nef gene codes for the negative factor (Nef) and is unique to genomes of the primate lentiviruses, the human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and the simian immunodeficiency virus (SIV). Nef is believed to play a crucial role in the pathogenesis of these viruses, and was shown to be necessary for the development of clinical AIDS in the simian model for the disease, i.e., in rhesus monkeys infected with an SIV strain carrying the wild-type (wt) nef gene. Despite numerous studies describing a role for the Nef in down-regulating CD4 receptor and interferring with normal transduction of T-cell signals in vitro and in vivo, little is known about the biochemistry and mechanism of action of this early viral regulatory protein. We previously identified conserved sequences within the Nef proteins that shared sequence or structural homologies with such functional domains as the nucleotide binding sites of the p60src and p21ras family of protein kinases, the dimerization domain of the leucine zipper class of proteins, and the minimal acidic activation domain of transcriptional activation factors. These conserved sequences could direct specific interactions between Nef and key cellular regulatory factors or function as sites critical for Nef action in vivo. Identification and characterization of the intracellular targets for Nef action and mapping their sites of interaction, could lead to an elucidation of the mechanism and function of Nef and facilitate development of drug strategies for blocking or attenuating its action. This report highlights results of studies that show that the zipper-like sequence in Nef mediates protein-protein interaction and is critical for homooligomerization in vitro. Similarly, data are presented to show that Nef and the signal transduction protein, c-Raf-1 kinase, interact in vitro presumably via a conserved acidic sequence motif at their carboxyl termini. The degree of variation in DNA sequence in the nef region was much less than that in the env region. While the differences between nef sequences collected over a 9 year span from a single patient also approached that seen between different individuals, the sequences were monophyletic as compared to representative database sequences. Thus, phylogenetic information is preserved for a longer period of time in the nef region as compared to the env region.